Team:Hannover/Design

iGEM Hannover 2020

Design

Summary: We first describe the biological design of our sensor. Secondly, we explain how we desgined a microfluidic measuring chamber to identify magnetized cells. Please follow us to Engineering to learn how we started to implement both concepts.

Biological design

We aimed to design a sensor that uses biological material to save the life of a patient who is dependent on his new implant and should not have the painful resulting from the formation of a biofilm.

All members of the team were committed to the development of a possible design of the sensor. We asked ourselves the following questions:

1. What must the sensor be able to do?

The sensor, which consists of encapsulated cells, must be able to perceive external influences and react to them in an appropriate way. Our cells must be able to react to a stimulation of bacterial toxins, which are released by bacteria in a biofilm. This should stimulate cellular signaling pathways that lead to the specific expression of reporter proteins. The sensor should work self-sufficiently: If stimuli are applied to the sensor, the sensor should correctly initiate signal cascades and reacts to the stimulus. Through these cellular processes the sensor should be able to indicate an infection in a selective way.

Figure 1: Our sensor reacts autonomously to the toxins of the biofilm. These lead to different signaling pathways in the cell, which result in a response that can be easily detected.

2. How should the stimulus lead to an answer?

The external stimuli should lead to the expression of two reporter proteins downstream of different promoters. The promoters must react to inflammatory molecules that are activated by signaling cascades induced by the bacterial toxins. The genetically modified cells in our project react to lipopolysaccharides or LPS for short. The NF-κB pathway is activated by this bacterial toxin [1]. LPS binds to the Toll-like receptor, which leads to activation of the IκBα kinase. This is able to phosphorylate IκBα, which means that it can no longer bind to NF-κB. The phosphorylation dissolves the connection, NF-κB is no longer bound and is able to translocate into the cell nucleus. Here it can initiate the expression of certain target genes by binding to different promoters and allowing downstream proteins to be strongly expressed. The signaling pathway is shown schematically in the figure below.

Figure 2: NF-κB-Pathway and activated signal cascades. Lipopolysaccharides bind to the Toll-like receptor, enabling the IκBα kinase to phosphorylate IκBα, which allows NF-κB to translocate into the nucleus to stimulate the expression of target genes.

The IL6-promotercreated part:
BBa_K3338008 has specific binding sites for certain inflammatory signals, such as NF-κB or AP-1. Exactly this promoter was chosen so that the activation of the IL6-promotercreated part:
BBa_K3338008 can occur via LPS, the toxin that is strongly produced by bacteria in biofilms. Via this LPS, signals are passed on, which should lead to the binding of transcription factors such as NF-κB to the IL6-promotercreated part:
BBa_K3338008 within our altered cells for the increased expression of our signal proteins. In addition to this study, a mutated IL6-promotercreated part:
BBa_K3338008 was used to adapt this promoter to the BioBrick system of the iGEM Foundation [2].

The next goal was to create two specially designed promoters. This had the aim to minimize the basal expression. That means that we only combined binding sites of AP-1 and NF-κB in our sequence to exclude all other influences by differential processes in the cell. All promoters are shown schematically in the following figure [3].

Figure 3: Used promoters. Besides the IL6-promotercreated part:
BBa_K3338008 and the CMV-promoterused part:
BBa_I712004, a mutated IL6-promoter (BioBrick format) and two self-designed primers were investigated. The self-designed primers consist of a different sequence of minimal CMV-promoterused part:
BBa_I712004, AP-1 and NF-κB binding sites to enable a response to LPS stimulation.

In order to push our project forward in a goal-oriented manner, we first tested all parts and constructs with a CMV-promoterused part:
BBa_I712004. This is due to the fact that the CMV-promoter does not react to LPS, or in general shows a very high basal expression in many cell types. For that reason, the CMV-promoter is the perfect way to verify the functionality and the correct localization of our transcripts and proteins [4].

3. What should the answer of our sensor look like?

Once the use and activation of certain promoters and signaling cascades had been clarified, the question of suitable signaling proteins arose. The first thing we came up with was the good idea of using the protein MagAcreated part:
BBa_K3338000. MagAcreated part:
BBa_K3338000 is a magnetizable transmembrane protein that acts as an iron transporter and performs important functions in different cell types. The great advantage of this protein in relation to our project is the fact that we can make the cells magnetizable via this protein [5]. This fact makes it possible to use the magnetizability of the implant by means of an MRI examination. This offers the immense possibility to examine the condition or the appearance of biofilm on implants with relatively low stress to the patient by means of a non-invasive imaging method. Our idea was therefore to examine the magnetization of the implant directly after the operation and to compare it with the magnetization after a certain period of time. By this examination method the basal expression/basal magnetization of the genetically modified cells can be compared with the magnetization at the time of the second measurement. If lower values than directly after the insertion of the implant can now be detected, it can be assumed that the cells have been degraded by the immune system and our sensor has therefore been destroyed. If, on the other hand, a strongly increased magnetization of the cells by the magnetosomes can be detected, the formation of a biofilm and the toxins associated with it is very likely. The principle of the use of MagAcreated part:
BBa_K3338000 in combination with a MRI examination is shown in the next illustration.

Figure 4: The used reporter protein MagAcreated part:
BBa_K3338000 and the possible visualization process via MRI examination. The transmembrane protein MagAcreated part:
BBa_K3338000 leads to the formation of magnetosomes, which allows the transfected cells to be visualized within an MRI investigation.

In order to further validate this principle, the secretion of a luciferase out of our cells is used to detect the biofilm in addition to this new technique. This can therefore be demonstrated in our in vitro experiments in the medium. In the cell-based sensor system the luciferase could be shown in blood and urine samples. The luciferase can be made visible by a simple assay using luminescence. Therefore, analogous to the MagAcreated part:
BBa_K3338000, the gene of Gaussia Luciferasecreated part:
BBa_K3338001 (hGluc) was cloned behind the different promoters. If a biofilm is formed, the luciferase is secreted and can be detected in blood and urine samples. The same principle of time-shifted measurement can be used as described above to differentiate the basal activity of the cells [6]. The principle of the luminescence measurement based on the Gaussia Luciferasecreated part:
BBa_K3338001 is shown in the next illustration.

Figure 5: Principle of detection via Gaussia Luciferasecreated part:
BBa_K3338001. After LPS stimulation, the luciferase is expressed and secreted into the medium. There it can be easily detected by a luminescence assay. In the context of the implant-associated sensor, luciferase can be detected in blood and urine samples.

After these two reporter proteins were determined for detection, the groundbreaking question of the possible combination and thus further validation of our system arose. For this purpose, we rely on an IREScreated part:
BBa_K3338004, an internal ribosome entry site and a P2Acreated part:
BBa_K3338003 peptide [7,8].

These different approaches are based on different biological expression processes. In an IREScreated part:
BBa_K3338004, the transcripts are translated separately, practically by two different ribosomes. IREScreated part:
BBa_K3338004 enables ribosomes to bind to a specific site on the RNA strand to initiate translation [9,10]. The first discovery of these sequences characterized by a specific secondary structure was found in viral genomes. IREScreated part:
BBa_K3338004 enables translation initiation independent of the 5'-cap of mRNA, which is normally required for ribosome binding and translation initiation [11]. The biology of an IREScreated part:
BBa_K3338004 is shown in the figure below.

Figure 6: Principle of simultaneous reporter protein expression via an internal ribosome entry site (IREScreated part:
BBa_K3338004). Via a secondary structure in the RNA, a ribosome can bind without a 5'-cap within the sequence under the control of a single promoter, thus creating the starting point for a second translation initiation [11].

Via P2Acreated part:
BBa_K3338003 sites, in contrast, a coherent transcript is generated, which is cut after the translation. It results in a linked primary transcript within a common open reading frame. This principle is shown in the next illustration. The exact mechanism of the P2Acreated part:
BBa_K3338003 site is not yet understood in detail [12,13]. It is believed that there is one peptide binding, which is prevented during translation, resulting in the formation of two separate proteins or peptides from one open reading frame during translation.

Figure 7: Principle of translation of two proteins via a P2Acreated part:
BBa_K3338003 sequence. The translation of a primary transcript from which the two individual proteins are separated is performed. The mechanism of translation and separation of the primary transcript via a P2Acreated part:
BBa_K3338003 site is not yet understood in detail.

Using these different approaches to combine different detection principles, our project will open up completely new possibilities for the non-invasive, early detection of biofilms. The patient is spared, both by simpler detection methods and by an earlier recognition of complications regarding the rejection of the implant.

Desing of a microfluidic measuring chamber for magnetized cells

The use of magnetic nanoparticles as biomarkers became standard practice in recent years. Their magnetic properties can be used for non-invasive imaging or highly specific treatment. For example, magnetic nanoparticles show a high potential for medical applications in diagnostics and cancer prohibition [14,15]. We aim to develop an early detection for biofilm adhesion on implant surfaces, minimizing the risk of more serious complications [16].

Our sensor cells use a special kind of magnetic nanoparticles, for the detection of biofilms, the so called magnetosomes. These are magnetic domains inside some cells (magnetotactic bacteria), which mainly consist of smaller magnetic particles build of magnetite crystals. The magnetosomes are present in the cell as chain-like structures. Magnetosomes show paramagnetic behavior. Under an external magnetic field, the magnetic dipole moments of the magnetite crystals align in parallel, forming a magnetic domain inside the cell.

We use this special property in our sensor cells to enable a non-invasive detection of biofilm adhesion on implant surfaces. The expression of the MagAcreated part:
BBa_K3338000 gene in the cells, results in the expression of the MagAcreated part:
BBa_K3338000 iron-channels on the cell membrane. Endogenous iron ions diffuse into the sensor cells, forming magnetosomes and making the sensor cells magnetizable. Non-invasive detection of the sensor-cells via MRI can therefor serve as an contrast agent of biofilm formation [14,17].

To characterize the magnetic properties of the cells, we designed a simple measuring chamber. With this measuring chamber, we are able to verify that our genetic construct is working. The easy to build and operate measuring chamber will also allow future iGEM Teams to characterize their own magnetic cells or particles in a fast and efficient way.

Main requirements for our chamber are simple utilization while obtaining a significant characterization of the magnetic particles and an easy manufacturing process. For manufacturing, 3D printing is the best option. Our design approach for the chamber is a sandwich structure, enabling the chamber to be printed with a low resolution printer. The chamber is designed using CAD-software Autodesk Inventor 2019. The printing template can be obtained by contacting us.

The chamber consists of two Y-channels connect through a mixing channel with respective inputs and outputs. The design was printed with a Formlabs 3 resin printer. A glass slip was added on the backside using Cyanoacrylate glue, making the chamber waterproof and allowing the flows be observed in real time.

Figure 8: 3D-computer-aided-design of our measuring chamber.

Bibliography and references

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